The present invention is generally in the field of receptors belonging to the TNF/NGF superfamily of receptors and the control of their biological functions. The TNF/NGF superfamily of receptors includes receptors such as the p55 and p75 tumor necrosis factor receptors (TNF-Rs) and the FAS ligand receptor (also called FAS/APO1 or FAS-R and hereinafter will be called FAS-R) and others. More specifically, the present invention concerns novel proteins which bind to the intracellular domains (IC) of the p55 and p75 TNF-Rs and the FAS-R, (these intracellular domains designated p55IC, p75IC and FAS-IC, respectively) and which novel proteins are capable of modulating the function of the p55 and p75 TNF-Rs and the FAS-R. One of the proteins capable of binding the p55IC of the intact p55-TNF-R is the p55IC itself in the form of a p55IC molecule or a portion thereof, such as for example, the so-called xe2x80x9cdeath domainxe2x80x9d (DD) of the p55IC. Thus, the present invention also concerns new TNF-associated effects that can be induced in cells in a ligand (TNF)-independent fashion by the intracellular domain of the p55 TNF-R (p55IC) or portions thereof. The present invention also concerns the preparation and uses of these novel p55 and p75 TNF-R-binding proteins, and FAS-R binding proteins, referred to herein as p55IC-, p75IC- and FAS-IC-binding proteins.
In another aspect, the present invention also concerns new soluble oligomeric TNF-Rs, oligomeric FAS-Rs and oligomeric receptors having a mixture of TNF-Rs and FAS-Rs, their uses, and methods for the production thereof.
Tumor Necrosis Factor (TNF-xcex1) and Lymphotoxin (TNF-xcex2) (hereinafter, TNF refers to both TNF-xcex1 and TNF-xcex2) are multifunctional pro-inflammatory cytokines formed mainly by mononuclear phagocytes, which have many effects on cells (Wallach, D. (1986) in: Interferon 7 (Ion Gresser, ed.), pp. 83-122, Academic Press, London; and Beutler and Cerami (1987)). Both TNF-xcex1 and TNF-xcex2 initiate their effects by binding to specific cell surface receptors. Some of the effects are likely to be beneficial to the organism: they may destroy, for example tumor cells or virus infected cells and augment antibacterial activities of granulocytes. In this way, TNF contributes to the defense of the organism against tumors and infectious agents and contributes to the recovery from injury. Thus, TNF can be used as an anti-tumor agent in which application it binds to its receptors on the surface of tumor cells and thereby initiates the events leading to the death of the tumor cells. TNF can also be used as an anti-infectious agent.
However, both TNF-xcex1 and TNF-xcex2 also have deleterious effects. There is evidence that over-production of TNF-xcex1 can play a major pathogenic role in several diseases. Thus, effects of TNF-xcex1, primarily on the vasculature, are now known to be a major cause for symptoms of septic shock (Tracey et al., 1986). In some diseases, TNF may cause excessive loss of weight (cachexia) by suppressing activities of adipocytes and by causing anorexia, and TNF-xcex1 was thus called cachectin. It was also described as a mediator of the damage to tissues in rheumatic diseases (Beutler and Cerami, 1987) and as a major mediator of the damage observed in graft-versus-host reactions (Piquet et al., 1987). In addition, TNF is known to be involved in the process of inflammation and in many other diseases.
Two distinct, independently expressed, receptors, the p55 and p75 TNF-Rs, which bind both TNF-xcex1 and TNF-xcex2 specifically, initiate and/or mediate the above noted biological effects of TNF. These two receptors have structurally dissimilar intracellular domains suggesting that they signal differently (See Hohmann et al., 1989; Engelmann et al., 1990; Brockhaus et al., 1990; Leotscher et al., 1990; Schall et al., 1990; Nophar et al., 1990; Smith et al., 1990; and Heller et al., 1990). However, the cellular mechanisms, for example, the various proteins and possibly other factors, which are involved in the intracellular signaling of the p55 an p75 TNF-Rs have yet to be elucidated (as set forth herein below, there is described for the first time, new proteins capable of binding to the p75IC and p55 IC). It is this intracellular signaling, which occurs usually after the binding of the ligand, i.e. TNF (xcex1 or xcex2), to the receptor, that is responsible for the commencement of the cascade of reactions that ultimately result in the observed response of the cell to TNF.
As regards the above mentioned cytocidal effect of TNF, in most cells studied so far, this effect is triggered mainly by the p55 TNF-R. Antibodies against the extracellular domain (ligand binding domain) of the p55 TNF-R can themselves trigger the cytocidal effect (see EP 412486) which correlates with the effectivity of receptor cross-linking by the antibodies, believed to be the first step in the generation of the intracellular signaling process. Further, mutational studies (Brakebusch et al., 1992; Tartaglia et al., 1993) have shown that the biological function of the p55 TNF-R depends on the integrity of its intracellular domain, and accordingly it has been suggested that the initiation of intracellular signaling leading to the cytocidal effect of TNF occurs as a consequence of the association of two or more intracellular domains of the p55 TNF-R. Moreover, TNF (xcex1 and xcex2) occurs as a homotrimer and as such has been suggested to induce intracellular signaling via the p55 TNF-R by way of its ability to bind to and to cross-link the receptor molecules, i.e. cause receptor aggregation. Herein below there is described how the p55IC and p55DD can self-associate and induce, in a ligand-independent fashion, TNF-associated effects in cells.
Another member of the TNF/NGF superfamily of receptors is the FAS receptor (FAS-R) which has also been called the FAS antigen, a cell-surface protein expressed in various tissues and sharing homology with a number of cell-surface receptors including TNF-R and NGF-R. The FAS-R mediates cell death in the form of apoptosis (Itoh et al., 1991), and appears to serve as a negative selector of autoreactive T cells, i.e. during maturation of T cells, FAS-R mediates the apoptotic death of T cells recognizing self-antigens. It has also been found that mutations in the FAS-R gene (lpr) cause a lymphoproliferation disorder in mice that resembles the human autoimmune disease systemic lupus erythematosus (SLE) (Watanabe-Fukunaga et al., 1992). The ligand for the FAS-R appears to be a cell-surface associated molecule carried by, amongst others, killer T cells (or cytotoxic T lymphocytesxe2x80x94CTLs), and hence when such CTLs contact cells carrying FAS-R, they are capable of inducing apoptotic cell death of the FAS-R-carrying cells. Further, a monoclonal antibody has been prepared that is specific for FAS-R, this monoclonal antibody being capable of inducing apoptotic cell death in cells carrying FAS-R, including mouse cells transformed by cDNA encoding human FAS-R (Itoh et al., 1991).
It has also been found that various other normal cells, besides T lymphocytes, express the FAS-R on their surface and can be killed by the triggering of this receptor. Uncontrolled induction of such a killing process is suspected to contribute to tissue damage in certain diseases, for example, the destruction of liver cells in acute hepatitis. Accordingly, finding ways to restrain the cytotoxic activity of FAS-R may have therapeutic potential.
Conversely, since it has also been found that certain malignant cells and HIV-infected cells carry the FAS-R on their surface, antibodies against FAS-R, or the FAS-R ligand, may be used to trigger the FAS-R mediated cytotoxic effects in these and thereby provide a means for combating such malignant cells or HIV-infected cells (see Itoh et al., 1991). Finding yet other ways for enhancing the cytotoxic activity of FAS-R may therefore also have therapeutic potential.
It has been a long felt need to provide a way for modulating the cellular response to TNF (xcex1 or xcex2) and FAS-R ligand, for example, in pathological situations as mentioned above, where TNF or FAS-R ligand is over-expressed it is desirable to inhibit the TNF- or FAS-R ligand-induced cytocidal effects, while in other situations, e.g. wound healing applications, it is desirable to enhance the TNF effect, or in the case of FAS-R, in tumor cells or HIV-infected cells it is desirable to enhance the FAS-R mediated effect.
A number of approaches have been made by the present inventors (see for example, European Application Nos. EP 186833, EP 308378, EP 398327 and EP 412486) to regulate the deleterious effects of TNF by inhibiting the binding of TNF to its receptors using anti-TNF antibodies or by using soluble TNF receptors (being essentially the soluble extracellular domains of the receptors) to compete with the binding of TNF to the cell surface-bound TNF-Rs. Further, on the basis that TNF-binding to its receptors is required for the TNF-induced cellular effects, approaches by the present inventors (see for example EPO 568925) have been made to modulate the TNF effect by modulating the activity of the TNF-Rs. Briefly, EPO 568925 relates to a method of modulating signal transduction and/or cleavage in TNF-Rs whereby peptides or other molecules may interact either with the receptor itself or with effector proteins interacting with the receptor, thus modulating the normal functioning of the TNF-Rs. In EPO 568925 there is described the construction and characterization of various mutant p55 TNF-Rs, having mutations in the extracellular, transmembranal, and intracellular domains of the p55 TNF-R. In this way regions within the above domains of the p55 TNF-R were identified as being essential to the functioning of the receptor, i.e. the binding of the ligand (TNF) and the subsequent signal transduction and intracellular signaling which ultimately results in the observed TNF-effect on the cells. Further, there is also described a number of approaches to isolate and identify proteins, peptides or other factors which are capable of binding to the various regions in the above domains of the TNF-R, which proteins, peptides and other factors may be involved in regulating or modulating the activity of the TNF-R. A number of approaches for isolating and cloning the DNA sequences encoding such proteins and peptides; for constructing expression vectors for the production of these proteins and peptides; and for the preparation of antibodies or fragments thereof which interact with the TNF-R or with the above proteins and peptides that bind various regions of the TNF-R, are also set forth in EPO 568925. However, no description is made in EPO 568925 of the actual proteins and peptides which bind to the intracellular domains of the TNF-Rs (e.g. p55 TNF-R), nor is any description made of the yeast two-hybrid approach to isolate and identify such proteins or peptides which bind to the intracellular domains of TNF-Rs. Similarly, heretofore there has been no disclosure of proteins or peptides capable of binding the intracellular domain of FAS-R.
Thus, when it is desired to inhibit the effect of TNF, or the FAS-R ligand, it would be desirable to decrease the amount or the activity of TNF-Rs or FAS-R at the cell surface, while an increase in the amount or the activity of TNF-Rs or FAS-R would be desired when an enhanced TNF or FAS-R ligand effect is sought. To this end the promoters of both the p55 TNF-R and the p75 TNF-R have recently been sequenced and analyzed by the present inventors and a number of key sequence motifs have been found that are specific to various transcription regulating factors, and as such the expression of these TNF-Rs can be controlled at their promoter level, i.e. inhibition of transcription from the promoters for a decrease in the number of receptors, and an enhancement of transcription from the promoters for an increase in the number of receptors (see WO 95/31206 and U.S. Ser. No. 08/600,203). Corresponding studies concerning the control of FAS-R at the level of the promoter of the FAS-R gene have yet to be reported.
Further, it should also be mentioned that, while it is known that the tumor necrosis factor (TNF) receptors, and the structurally-related receptor FAS-R, trigger in cells, upon stimulation by leukocyte-produced ligands, destructive activities that lead to their own demise, the mechanisms of this triggering are still little understood. Mutational studies indicate that in FAS-R and the p55 TNF receptor (p55-R) signaling for cytotoxicity involve distinct regions within their intracellular domains (Brakebusch et al., 1992; Tartaglia et al., 1993; Itoh and Nagata, 1993). These regions (the xe2x80x9cdeath domainsxe2x80x9d) have sequence similarity. The xe2x80x9cdeath domainsxe2x80x9d of both FAS-R and p55-R tend to self-associate. Their self-association apparently promotes that receptor aggregation which is necessary for initiation of signaling (as set forth herein below, as well as Song et al., 1994; Wallach et al., 1994; Boldin et al., 1995) and at high levels of receptor expression can result in triggering of ligand-independent signaling (as set forth herein below, and Boldin et al., 1995).
Thus, prior to the present invention, there have not been provided proteins which may regulate the effect of ligands belonging to the TNF/NGF superfamily, such as the TNF or FAS-R ligand effect on cells, by mediation of the intracellular signaling process, which signaling is probably governed to a large extent by the intracellular domains (ICs) of the receptors belonging to the TNF/NGF superfamily of receptors, such as those of the TNF-Rs, i.e. the p55 and p75 TNF-R intracellular domains (p55IC and p75IC, respectively), as well as the FAS-IC.
Accordingly, it is one aim of the invention to provide proteins which are capable of binding to the intracellular domains of the TNF-Rs and FAS-R, which proteins are presently believed to be involved in the intracellular signaling process initiated by the binding of TNF to its receptors, or the binding of FAS ligand to its receptor.
Another aim of the invention is to provide antagonists (e.g. antibodies) to these intracellular domain-binding proteins (IC-binding proteins) which may be used to inhibit the signaling process, when desired, when such IC-binding proteins are positive signal effectors (i.e. induce signaling), or to enhance the signaling process, when desired, when such IC-binding proteins are negative signal effectors (i.e. inhibit signaling).
Yet another aim of the invention is to use such IC-binding proteins to isolate and characterize additional proteins or factors, which may, for example, be involved further downstream in the signaling process, and/or to isolate and identify other receptors further upstream in the signaling process to which these IC-binding proteins bind (e.g. other TNF-Rs or related receptors), and hence, in whose function the IC-binding proteins are also involved.
Moreover, it is an aim of the present invention to use the above-mentioned IC-binding proteins as antigens for the preparation of polyclonal and/or monoclonal antibodies thereto. The antibodies, in turn, may be used for the purification of the new IC-binding proteins from different sources, such as cell extracts or transformed cell lines.
Furthermore, these antibodies may be used for diagnostic purposes, e.g. for identifying disorders related to abnormal functioning of cellular effects mediated by receptors belonging to the TNF/NGF receptor superfamily.
A further aim of the invention is to provide pharmaceutical compositions comprising the above IC-binding proteins, and pharmaceutical compositions comprising the IC-binding protein antagonists, for the treatment or prophylaxis of TNF-induced or FAS ligand-induced conditions, for example, such compositions can be used to enhance the TNF or FAS ligand effect or to inhibit the TNF or FAS ligand effect depending on the above noted nature of the IC-binding protein or antagonist thereof contained in the composition.
Moreover, in accordance with another aim of the present invention, there is disclosed other ways for eliminating or antagonizing endogenously formed or exogenously administered TNF or FAS-R ligand, by the use of soluble oligomeric TNF-Rs, oligomeric FAS-Rs, or oligomers being a mixture of TNF-Rs and FAS-Rs. In this respect it should be mentioned that one attempt in this direction was the isolation and recombinant production of a TNF Binding Protein called TBP-I which was shown to be able to antagonize the effects of TNF. This antagonism was determined both by measuring reduction of the cytotoxic activity of TNF, as well as by measuring interference of TNF binding to its receptors (EP 308 378). TBP-I was shown to protect cells from TNF toxicity at concentrations of a few nanograms per ml and to interfere with the binding of both TNF-xcex1 and TNF-xcex2 to cells, when applied simultaneously with these cytokines. Further examination of the mechanism by which TBP-I functions revealed that TBP-I does not interact with the target cell, but rather blocks the function of TNF by binding TNF specifically, thus competing for TNF with the TNF receptor.
Consequently, with a different purification technique, the presence of two active components was found: one, TBP-I, and also a second TNF-binding protein which we called TBP-II (first described in EP 398327). Both proteins provide protection against the in vitro cytocidal effect of TNF and both bind TNF-xcex2 less effectively than TNF-xcex1. Although in SDS PAGE analysis the two proteins, TBP-I and TBP-II, appeared to have a very similar molecular size, they could clearly be distinguished from each other by lack of immunological cross reactivity, differing N-terminal amino acid sequences and differing amino acid composition.
However, the above noted earlier soluble TNF binding proteins are monomeric and being capable of binding only one monomer of the TNF homotrimer, the natural ligand, which still permits TNF activity (i.e. incomplete neutralization) by virtue of the TNF still having two active monomers unbound by the TNF binding proteins. Further, heretofore there has been no disclosure of soluble FAS-Rs (soluble FAS-R ligand binding proteins) capable of binding to FAS-R ligand which is known to be a homotrimeric, cell-surface associated molecule.
A so-called xe2x80x9cdeath domainxe2x80x9d of the p55-IC (Tartaglia et al., 1993) has been disclosed, but did not show, in accordance with the present invention, that the p55-IC and the xe2x80x9cdeath domainxe2x80x9d thereof self-associates, this self-association being primarily responsible for the signaling leading to induction of cell cytotoxis. Moreover, this publication is silent on the possibility of producing the soluble, oligomeric TNF-Rs, or the soluble, oligomeric FAS-Rs, or mixed oligomeric thereof, nor does it disclose other TNF-associated effects induced by the p55-IC or portions thereof, e.g. IL-8 gene expression induction, all of the present invention. Likewise, another publication, published after the date of the present invention, disclosed the aggregation (i.e. self-association) ability of the p55-IC, but did not relate, as noted above, to the usage thereof to prepare soluble, oligomeric TNF-Rs or FAS-Rs nor to the other TNF-associated effects induced in a ligand-independent manner by the p55-IC or portions thereof according to the invention.
Citation of any document herein is not intended as an admission that such document is pertinent prior art, or considered material to the patentability of any claim of the present application. Any statement as to content or a date of any document is based on the information available to applicant at the time of filing and does not constitute an admission as to the correctness of such a statement.
In accordance with the present invention, we have found novel proteins which are capable of binding to either the intracellular domain of the p55 TNF-R (the p55IC-binding proteins), of the p75 TNF-R (the p75IC-binding proteins), and of the FAS-R (the FAS-IC-binding proteins). These p55IC-, p75IC- and FAS-IC-binding proteins may act as mediators or modulators of the TNF or FAS-R ligand effect on cells by way of mediating or modulating the intracellular signaling process which usually occurs following the binding of TNF to the p55 and/or p75 TNF-R, or the binding of the FAS-R ligand at the cell surface. Further, it has been surprisingly and unexpectedly found that the p55IC and FAS-IC are capable of self association and that fragments of the p55IC and FAS-IC are similarly capable of binding to the p55 IC, particularly the so-called xe2x80x9cdeath domainsxe2x80x9d (DD) within the ICs of these receptors, i.e. the p55DD and FAS-DD. Thus, p55 IC and FAS-IC and their fragments also represent proteins capable of binding to the p55IC and FAS-IC and hence may be modulators of the TNF or FAS-R ligand effect on cells.
Furthermore, the nature of the binding of one of the novel proteins of the invention, the herein designated 55.11 protein, to the intracellular domain of p55-TNF-R has been more fully elucidated (see Example 1).
Moreover, in another aspect, the present invention is based on the finding that the intracellular domain of the p55 TNF receptor (p55-IC), a region contained therein, the so-called p55-IC xe2x80x9cdeath domainxe2x80x9d, the intracellular domain of the FAS/APO1 receptor (FAS-IC), and a region contained therein, the so-called FAS-IC xe2x80x9cdeath domainxe2x80x9d are capable of self-association. Accordingly, it is possible to construct by standard recombinant DNA techniques, a soluble, oligomeric TNF receptor being a fusion product, containing at least two extracellular domains of a TNF receptor at its one end, and at its other end at least two of the above noted self-associating intracellular domains or portions thereof, which self-associate to provide an oligomer having at least two such fusion products linked together. Such a soluble, oligomeric TNF-R is thus capable of binding two monomers of the naturally-occurring TNF homotrimer, and as such effectively neutralizes TNF activity. The neutralization of TNF activity being desirable in all of the above mentioned conditions wherein TNF is overproduced endogenously or is administered exogenously in high doses resulting in undesirable side effects. Further, the effective binding of TNF by the soluble, oligomeric receptors of the invention may also serve to allow for the binding of exogenously added TNF and its subsequent desired slow-release in conditions where TNF is administered for its beneficial effects, e.g. in tumor therapy. Likewise, it is also possible to construct by standard recombinant DNA techniques an oligomeric FAS-R being a fusion product, containing at least two extracellular domains of a FAS-R at its one end, and at its other end at least two of the above noted self-associating intracellular domains or portions thereof, which self-associate to provide an oligomer having at least two such fusion products linked together. Such an oligomeric FAS-R is thus capable of binding two monomers of the naturally occurring FAS-R ligand homotrimer, and as such effectively neutralizes FAS-R ligand activity. The neutralization of FAS-R ligand activity being desirable in all of the above mentioned conditions where excess amounts thereof are associated with undesirable side effects. In a similar fashion, and in view of recent reports indicating a possible associating between TNF and FAS-R ligand-induced effects on cells and hence also a possible association, geographically at the cell surface where they attach to their receptors, it is also possible to construct by standard recombinant DNA techniques a mixed oligomeric receptor having specificity for both TNF and FAS-R ligand. Such a mixed oligomer would be a mixture of the above noted fusion products containing at least one extracellular domain of a TNF-R and at least one extracellular domain of a FAS-R at its one end, and at its other end at least two of the above mentioned self-associating intracellular domains or portions thereof, which self-associate to provide a mixed oligomer having at least two such fusion products linked together. Such a mixed oligomer is thus capable of binding at least one monomer of TNF and one monomer of FAS-R ligand at the same time, thereby reducing or effectively neutralizing the TNF and FAS-R ligand activities at the cell surface in conditions, as noted above where excess amounts of these two cytokines are associated with undesirable cellular effects. As noted above, the FAS-R ligand is usually cell-surface-associated, and recent reports also describe cell-surface-associated forms of TNF. Hence, these mixed TNF-R/FAS-R oligomers are especially useful for neutralization of TNF and FAS-R ligand activities at the cell surface.
Accordingly, the present invention provides a DNA sequence encoding a protein capable of binding to one or more of the intracellular domains of one or more receptors belonging to the tumor necrosis factor/nerve growth factor (TNF/NGF) superfamily of receptors.
In particular, the present invention provides a DNA sequence selected from the group consisting of:
(a) a cDNA sequence derived from the coding region of a native TNF-R intracellular domain-binding protein;
(b) DNA sequences capable of hybridization to a DNA of (a) under moderately stringent conditions and which encode a biologically active TNF-R intracellular domain-binding protein; and
(c) DNA sequences which are degenerate as a result of the genetic code to the DNA sequences defined in (a) and (b) and which encode a biologically active TNF-R intracellular domain-binding protein.
The present invention also provides a DNA sequence selected from the group consisting of:
(a) a cDNA sequence derived from the coding region of a native FAS-R intracellular domain-binding protein;
(b) DNA sequences capable of hybridization to a cDNA of (a) under moderately stringent conditions and which encode a biologically active FAS-R intracellular domain-binding protein; and
(c) DNA sequences which are degenerate as a result of the genetic code to the DNA sequences defined in (a) and (b) and which encode a biologically active FAS-R intracellular domain-binding protein.
In embodiments of the present invention the DNA sequences encode p55 TNF-R, p75 TNF-R and FAS-R intracellular domain-binding proteins, such as those encoding the herein designated proteins 55.1, 55.3, 55.11, 75.3, 75.16, F2, F9, DD11, E3, E15, E19, 230, 4, 65, 14v1 and 16v1.
The present invention also provides a protein or analogs or derivatives thereof encoded by any of the above sequences of the invention, said proteins, analogs and derivatives being capable of binding to one or more of the intracellular domains of one or more TNF-Rs or FAS-R. Embodiments of this aspect of the invention include the herein designated proteins 55.1, 55.3, 55.11, 75.3, 75.16, F2, F9, DD11, E3, E15, E19 and 230, their analogs and their derivatives as well as the p55IC-binding proteins encoded by the clones designated 4, 65, 14v1 and 16v1.
Also provided by the present invention are vectors encoding the above proteins of the invention, which contain the above DNA sequences of the invention, these vectors being capable of being expressed in suitable eukaryotic or prokaryotic host cells; transformed eukaryotic or prokaryotic host cells containing such vectors; and a method for producing the proteins, analogs or derivatives of the invention by growing such transformed host cells under conditions suitable for the expression of said protein, analogs or derivatives, effecting post-translational modifications of said protein as necessary for obtention of said protein and extracting said expressed protein, analogs or derivatives from the culture medium of said transformed cells or from cell extracts of said transformed cells.
In another aspect, the present invention also provides antibodies or active derivatives or fragments thereof specific to the proteins, analogs and derivatives thereof, of the invention.
By yet another aspect of the invention, there are provided various uses of the above DNA sequences or the proteins which they encode, according to the invention, which uses include amongst others:
(i) a method for the modulation of the TNF or FAS-R ligand effect on cells carrying a TNF-R or a FAS-R, comprising treating said cells with one or more proteins, analogs or derivatives selected from the group consisting of the proteins, analogs and derivatives, according to the invention, and a protein being the p55IC, p55DD, FAS-IC or FAS-DD, analogs or derivatives thereof, all of said proteins being capable of binding to the intracellular domain and modulating the activity of said TNF-R or FAS-R, wherein said treating of the cells comprises introducing into said cells said one or more proteins, analogs or derivatives in a form suitable for intracellular administration or introducing into said cells, in the form of a suitable expression vector, the DNA sequence encoding said one or more proteins, analogs or derivatives;
(ii) a method for modulating the TNF or FAS-R ligand effect on cells carrying a TNF-R or a FAS-R comprising treating said cells with antibodies or active derivatives or fragments thereof according to the invention;
(iii) a method for modulating the TNF or FAS-R ligand effect on cells carrying a TNF-R or FAS-R comprising treating said cells with an oligonucleotide sequence encoding an antisense sequence of at least part of the sequence according to the invention, or encoding an antisense sequence of the p55IC, p55DD, FAS-IC, or FAS-DD sequence, said oligonucleotide sequence being capable of blocking the expression of at least one of the TNF-R or FAS-R intracellular domain binding proteins;
(iv) a method for modulating the TNF or FAS-R ligand effect on cells carrying a TNF-R or FAS-R comprising:
(a) constructing a recombinant animal virus vector carrying a sequence encoding a viral surface protein that is capable of binding to a specific cell surface receptor and a sequence selected from an oligonucleotide sequence encoding an antisense sequence of at least part of the sequence according to the invention and an oligonucleotide sequence encoding an antisense sequence of the p55IC, p55DD, FAS-IC, or FAS-DD sequence, said oligonucleotide sequence being capable of blocking the expression of at least one of the TNF-R or FAS-R intracellular domain binding proteins when introduced into said cells by said virus; and
(b) infecting said cells with said vector of (a).
(v) a method for modulating the TNF or FAS-R ligand effect on cells carrying a TNF-R or a FAS-R, comprising treating said cells with a suitable vector encoding a ribozyme having a sequence specific to a sequence selected from an mRNA sequence encoding a protein, analog or derivative of the invention and an mRNA sequence encoding the p55IC, p55DD, FAS-IC or FAS-DD, said ribozyme sequence capable of interacting with said mRNA sequence and capable of cleaving said mRNA sequence resulting in the inhibition of the expression of the protein, analog or derivative of the invention or of the expression of the p55IC, p55DD, FAS-IC or FAS-DD;
(vi) a method for treating tumor cells or HIV-infected cells, or other diseased cells, comprising:
(a) constructing a recombinant animal virus vector carrying a sequence encoding a viral surface protein that is capable of binding to a tumor cell surface receptor or HIV-infected cell surface receptor or is capable of binding to another cell surface receptor of other diseased cells and a sequence selected from a sequence according to the invention encoding a protein, analog or derivative of the invention and a sequence encoding the p55IC, p55DD, FAS-IC, FAS-DD, or a biologically active analog or derivative thereof, said protein, analog or derivative of the invention, p55IC, p55DD, FAS-IC, FAS-DD, analog or derivative, when expressed in said tumor cell or HIV-infected cell, or other diseased cell being capable of killing said cell; and
(b) infecting said tumor cells or HIV-infected cells or other infected cells with said vector of (a);
(vii) a method for isolating and identifying proteins, factors or receptors capable of binding to the intracellular domain binding proteins according to the invention, comprising applying the procedure of affinity chromatography in which said protein according to the invention is attached to the affinity chromatography matrix, said attached protein is brought into contact with a cell extract and proteins, factors or receptors from cell extract which bound to said attached protein are then eluted, isolated analyzed;
(viii) a method for isolating and identifying proteins, capable of binding to the intracellular domain binding proteins according to the invention, comprising applying the yeast two-hybrid procedure in which a sequence encoding said intracellular domain binding protein is carried by one hybrid vector and a sequence from a cDNA or genomic DNA library is carried by the second hybrid vector, the vectors then being used to transform yeast host cells and the positive transformed cells being isolated, followed by extraction of the said second hybrid vector to obtain a sequence encoding a protein which binds to said intracellular domain binding protein; and
(ix) a method for isolating and identifying a protein capable of binding to the intracellular domains of TNF-Rs or FAS-R comprising applying the procedure of non-stringent southern hybridization followed by PCR cloning, in which a sequence or parts thereof according to the invention is used as a probe to bind sequences from a cDNA or genomic DNA library, having at least partial homology thereto, said bound sequences then amplified and cloned by the PCR procedure to yield clones encoding proteins having at least partial homology to said sequences according to the invention.
The present invention also provides a pharmaceutical composition for the modulation of the TNF- or FAS ligand-effect on cells comprising, as active ingredient, any one of the following: (i) a protein according to the invention, or the protein p55IC, p55DD, FAS-IC or FAS-DD, its biologically active fragments, analogs, derivatives or mixtures thereof; (ii) a recombinant animal virus vector encoding a viral surface protein capable of binding to a TNF-R or FAS-Rxe2x80x94carrying cellxe2x80x94or tumor cell-specific receptor and a sequence encoding a protein, analog or derivative of the invention or encoding the p55IC, p55DD, FAS-IC or FAS-DD; (iii) a recombinant animal virus vector encoding a viral surface protein as in (ii) above and an oligonucleotide sequence encoding an antisense sequence of the p55IC, p55DD, FAS-IC or FAS-DD sequence; and (iv) a vector encoding a ribozyme of sequence capable of interacting with a mRNA sequence encoding a protein, analog or derivative of the invention or a mRNA sequence encoding the p55IC, p55DD, FAS-IC or FAS-DD.
A specific embodiment of the above aspects of the invention is the use of the p55-IC or DNA encoding therefor. This embodiment is based on the discovery that the p55-IC may in a ligand (TNF)-independent fashion induce other TNF-associated effects in cells. Accordingly, there is provided a method for inducing TNF-associated effects in cells or tissues comprising treating said cells with one or more proteins, analogs or derivatives thereof, said one or more proteins being selected from a protein being essentially all of the self-associating intracellular domain of the p55 TNF-R (p55-IC) or portions thereof capable of self-associating and inducing, in a ligand (TNF)-independent manner, said TNF effect in the cells, wherein said treating of the cells comprises introducing into said cells said one or more proteins, analogs or derivatives in a form suitable for intracellular introduction thereof, or introducing into said cells a DNA sequence encoding said one or more proteins, analogs or derivatives in the form of a suitable vector carrying said sequence, said vector being capable of effecting the insertion of said sequence into said cells in a way that said sequence is expressed in said cells.
Embodiments of the above method of the invention include:
(i) a method wherein said treating of cells is by transfection of said cells with a recombinant animal virus vector comprising the steps of:
(a) constructing a recombinant animal virus vector carrying a sequence encoding a viral surface protein (ligand) that is capable of binding to a specific cell surface receptor on the surface of said cells to be treated, and a second sequence encoding a protein being the p55-IC, portions thereof, analogs and derivatives of all of the foregoing, said protein when expressed in said cells being capable of self-association and induction of said one or more TNF-associated effects; and
(b) infecting said cells with the vector of (a).
(ii) a method wherein said TNF effect to be induced in said cells is the induction of IL-8 gene expression, said vector carrying a sequence encoding essentially all of said p55-IC, portions thereof, analogs and derivatives of all of the foregoing, which are capable, when expressed in the cells of self-association and signaling for the induction of said IL-8 gene expression.
(iii) a method for treating tumor cells or virally-infected cells, or for augmenting the antibacterial effect of granulocytes, wherein said viral vector carries a sequence encoding a viral ligand capable of binding a specific cell surface receptor on the surface of said tumor cells, virally-infected cells or granulocytes and a sequence encoding said p55-IC portions thereof, analogs and derivatives thereof, which when expressed in said tumor, virally-infected or granulocyte cells induces TNF-associated effects leading to the death of these cells.
(iv) a method for treating tumor cells, wherein said p55-IC, portions thereof, analogs or derivatives thereof, when expressed in the tumor cells, induce the expression of IL-8 which leads to the killing of said tumor cells by its chemotactic activity which attracts granulocytes and other lymphocytes to the tumor cells resulting in the death of the tumor cells.
In this aspect of the invention, there is thus also provided the intracellular domain of the p55-R (p55-IC), portions, analogs and derivatives of all of the aforegoing for use in the treatment of cells by induction therein of TNF-associated effects; and the following embodiments thereof:
(i) the p55-IC, portions, analogs and derivatives for use in the treatment of cells by induction therein of IL-8 gene expression;
(ii) the p55-IC, portions, analogs and derivatives for use in the treatment of tumor cells by induction therein of IL-8 gene expression resulting in the killing of the tumor cells.
Moreover, in this aspect of the invention there is provided a pharmaceutical composition for treating cells by induction therein of TNF-associated effects, comprising, as active ingredient, p55-IC, portions thereof, analogs and derivatives of all of the aforegoing, and a pharmaceutically acceptable carrier; and the following embodiments thereof:
(i) a pharmaceutical composition for treating cells by induction therein of TNF-associated effects, comprising, as active ingredient a recombinant animal virus vector encoding p55-IC, portions thereof, analogs and derivatives of all of the aforegoing, and a protein capable of binding a cell surface protein on the cells to be treated;
(ii) a pharmaceutical composition for the treatment of tumor cells, administration of said composition leading to the induction of IL-8 expression, and subsequent killing of the tumor cells.
As yet another aspect, the present invention provides a soluble, oligomeric tumor necrosis factor receptor (TNF-R) comprising at least two self-associated fusion proteins, each fusion protein having (a) at its one end, a TNF binding domain selected from the extracellular domain of a TNF-R, analogs or derivatives thereof, said extracellular domain, analogs or derivatives thereof being incapable of deleterious self-association and being able to bind TNF; and (b) at its other end, a self-associating domain selected from (i) essentially all of the intracellular domain of the p55 TNF-R (p55-IC), extending from about amino acid residue 206 to about amino acid residue 426 of the native p55 TNF-R molecule (p55-R, SEQ ID NO:37); (ii) the death domain of the p55-IC extending from about amino acid residue 328 to about amino acid residue 426 of the native p55-R (SEQ ID NO:37); (iii) essentially all of the intracellular domain of the FAS/APO1 receptor (FAS-IC); (iv) the death domain of FAS-IC; and (v) analogs, fractions or derivatives of any one of (i)-(iv) being capable of self-association, wherein said at least two self-associated proteins self-associate only at said ends (b) having said ends (a) capable of binding to at least two TNF monomers, each end (a) capable of binding one TNF monomer; and salts and functional derivatives of said soluble, oligomeric TNF-R.
Embodiments of this aspect of the invention include all of the above combinations of ends (a) with ends (b) as defined above, for example, a soluble, oligomeric TNF-R comprising as extracellular domain, the p55-R extracellular domain and as self-associating intracellular domain, the p55-IC.
Moreover, there is also provided a process for producing the soluble oligomeric TNF-R of the invention comprising:
(a) the construction of an expression vector encoding any one of said fusion proteins, the DNA sequence of each of said ends of the fusion protein being obtained from cloned DNA sequences encoding essentially all of said extracellular domain of the TNF-R, analogs or derivatives thereof; and from cloned DNA sequences encoding essentially all of said p55-IC, p55-IC death domain, FAS-IC, FAS-IC death domain, analogs or derivatives of all of the aforegoing, said ends being ligated together to form a fusion protein sequence, and said fusion protein sequence being inserted into said vector under the control of transcriptional and translational regulatory sequences;
(b) introduction of the vector of (a) into a suitable host cell in which said fusion protein is expressed; and
(c) purification of the fusion protein expressed in said host cells, said fusion protein self-associating prior to, during, or following the purification process to yield a soluble, oligomeric TNF-R.
Furthermore, there is also provided a vector encoding the above fusion proteins, useful in the above method of the invention; host cells containing the vector; as well as a pharmaceutical composition comprising the soluble, oligomeric TNF-R, salts or functional derivatives thereof and mixtures of any of the aforegoing according to the invention, as active ingredient, together with a pharmaceutically acceptable carrier. Similarly, the soluble, oligomeric TNF-R, salts, functional derivatives thereof and mixtures of any of the aforegoing, according to the invention, are provided for use in antagonizing the deleterious effect of TNF in mammals, especially in the treatment of conditions wherein an excess of TNF is formed endogenously or is exogenously administered; or alternatively, for use in maintaining prolonged beneficial effects of TNF in mammals when used with TNF exogenously administered.
Along the lines set forth concerning the above aspect of the invention, it has also been discovered that it is possible to construct a soluble, oligomeric FAS/APO1 receptor (FAS-R) which is useful for antagonizing the deleterious effects of the FAS ligand. Accordingly, in a further aspect, the present invention provides a soluble, oligomeric FAS/APO1 receptor (FAS-R) comprising at least two self-associated fusion proteins, each fusion protein having (a) at its one end, a FAS ligand binding domain selected from the extracellular domain of a FAS-R, analogs or derivatives thereof being incapable of self-associating and being able to bind FAS ligand; and (b) at its other end, a self-associating domain selected from (i) essentially all of the intracellular domain of the p55 TNF-R (p55-IC), extending from about amino acid residue 206 to about amino acid residue 426 of the native p55 TNF-R molecule (p55-R; SEQ ID NO:37); (ii) the death domain of the p55-IC extending from about amino acid residue 328 to about amino acid residue 426 of the native p55-R (SEQ ID NO:37); (iii) essentially all of the intracellular domain of the FAS/APO1 receptor (FAS-IC); (iv) the death domain of FAS-IC; and (v) analogs or derivatives of any one of (i)-(iv) being capable of self-association, wherein said at least two self-associated proteins only self-associate at said ends (b) having said ends (a) capable of binding to at least two FAS ligand monomers, each end (a) capable of binding one FAS ligand monomer; and salts and functional derivatives of said soluble, oligomeric FAS-R.
In accordance with this aspect of the invention, there is also provided a process for the production of the soluble, oligomeric FAS-R comprising:
(a) the construction of an expression vector encoding any one of said fusion proteins, the DNA sequence of each of said ends of the fusion protein being obtained from cloned DNA sequences encoding essentially all of said extracellular domain of the FAS-R, analogs or derivatives thereof; and from cloned DNA sequences encoding essentially all of said p55-IC, p55-IC death domain, FAS-IC, FAS-IC death domain, analogs or derivatives thereof of all the aforegoing, said ends being ligated together to form a fusion protein sequence, and said fusion protein sequence being inserted into said vector under the control of transcriptional and translational regulatory sequences;
(b) introduction of the vector of (a) into a suitable host cell in which said fusion protein is expressed; and
(c) purification of the fusion protein expressed in the host cells, said fusion protein self-associating prior to, during, or following the purification process to yield a soluble, oligomeric FAS-R.
Moreover, also provided are an expression vector containing the fusion protein sequence encoding the soluble oligomeric FAS-R, useful in the above process; host cells containing the vector; and pharmaceutical compositions comprising the soluble, oligomeric FAS-R, salts or functional derivatives thereof or mixtures of any of the aforegoing as active ingredient together with a pharmaceutically acceptable carrier. Similarly, there is provided a soluble, oligomeric FAS-R, salts or functional derivatives thereof or mixtures of any of the aforegoing, for use in antagonizing the deleterious effect of FAS ligand in mammals, especially in the treatment of conditions wherein an excess of the FAS ligand is formed endogenously or is exogenously administered.
In a similar fashion to that noted above concerning the oligomeric TNF-Rs and oligomeric FAS-Rs, it is also possible to prepare mixed oligomers having binding specificity for both TNF and FAS-R ligand. Thus, the present invention also provides a mixed oligomeric TNF-R/FAS-R comprising at least two self-associated fusion proteins, one of which fusion proteins is selected from any one of the above mentioned TNF-specific fusion proteins, and the other fusion protein is selected from any one of the above mentioned FAS-R ligand-specific fusion proteins, to provide a mixed oligomer having at least one TNF-R extracellular domain and at least one FAS-R extracellular domain associated by virtue of the self-association between the intracellular domains or portions thereof fused to each of these extracellular domains. These mixed oligomeric receptors are prepared by preparing, as noted above, the oligomeric TNF-Rs and the oligomeric FAS-Rs and then mixing these together and subsequently selecting, by standard procedures, those oligomers having binding specificity for both FAS-R ligand and TNF. Another way for preparing the mixed oligomeric receptors is by co-transfecting suitable host cells with vectors, as noted above, encoding any of the TNF-specific fusion proteins (soluble TNF-Rs) and encoding any of the FAS-R ligand-specific fusion proteins (soluble FAS-Rs), purifying the expressed fusion proteins which self-associate prior to, during, or following the purification to yield oligomeric receptors, and then selecting by standard procedures, those oligomeric receptors which are capable of binding to both TNF and FAS-R ligand.
Likewise, there is also provided pharmaceutical compositions comprising the mixed oligomeric receptors, salts or functional derivatives thereof or mixtures of any of the aforegoing as active ingredient together with a pharmaceutically acceptable carrier. In addition, there is provided the mixed oligomeric receptors, salts or functional derivatives thereof or mixtures of any of the aforegoing, for use in antagonizing the deleterious effects of both TNF and FAS-R ligand in mammals, especially in the treatment of conditions wherein an excess of TNF and FAS-R ligand is formed endogenously or is exogenously administered; or alternatively, for use in maintaining prolonged (slow-release) beneficial effects of TNF and/or FAS-R ligand in mammals when used with TNF and/or FAS-R ligand (in soluble form) exogenously administered.
Other aspects and embodiments of the present invention are also provided as arising from the following detailed description of the invention.
It should be noted that, where used throughout, the following terms: xe2x80x9cModulation of the TNF-effect on cellsxe2x80x9d and xe2x80x9cModulation of the FAS-ligand effect on cellsxe2x80x9d are understood to encompass in vitro as well as in vivo treatment.